Inflammatory airways diseases - asthma, chronic bronchitis and cystic fibrosis - remain important causes of morbidity and mortality in the U.S. The purpose of this application is to study the effects of the dietary flavonol quercetin, an antioxidant and inhibitor of phosphatidylinositol (PI) 3-kinase, on airway inflammation. We will examine the effects of quercetin on biochemical signaling pathways regulating innate immune responses in cultured airway epithelial cells, and on two mouse models of airways disease, cockroach allergen sensitization (mimicking asthma) and Pseudomonas endobronchial infection (mimicking cystic fibrosis and chronic bronchitis). Our pilot data suggest that quercetin attenuates airway epithelial cell expression of inter- leukin (IL)-8, a neutrophil chemoattractant, and granulocyte macrophage-colony stimulating factor (GM- CSF), an eosinophil chemoattractant, in response to pro-inflammatory stimuli. We propose the general hypothesis that quercetin blocks airway inflammation by attenuating epithelial cell chemoattractant expression via both PI 3-kinase-dependent and -independent mechanisms. To test this, we propose the following Aims: Specific Aim 1. Examine the effects of quercetin on airway epithelial cell signaling pathways regulating the innate immune response to pro-inflammatory stimuli. We will measure airway epithelial cell IL-8 and GM-CSF expression in response to stimuli relevant to asthma, cystic fibrosis and chronic bronchitis - TNFalpha, cockroach extract and Pseudomonas aeruginosa. 16HBE14o- human airway epithelial cells will be studied, with key experiments repeated in primary cells. IL-8 and GM-CSF expression will be assessed by ELISA and reporter assays. The requirement of PI 3-kinase activation, Akt and intracellular H2O2 generation will be assessed using chemical and dominant-negative inhibitors. The effects of quercetin on IL- 8 and GM-CSF expression, PI 3-kinase and Akt activation, and H2O2 generation will be measured. Specific Aim 2. Examine the anti-inflammatory effects of quercetin in two mouse models of inflammatory airways diseases. We will pre-treat cockroach allergen-sensitized and challenged mice, as well as mice infected with Pseudomonas aeruginosa in the airways, with quercetin or vehicle. Airway inflammation will be assessed by histological methods and cell counts. Airway chemokine expression will be assessed by ELISA and immunohistochemistry. Akt phosphorylation and activation will be assessed by immunohistochemistry and in vitro kinase assay. Airways responsiveness will be measured by methacholine challenge testing. Blood and lung quercetin levels will be assessed by HPLC with electrochemical detection. This project examines the value of quercetin, a plant pigment, in preventing airway inflammation in mouse models of disease. This work may lead to new treatments for asthma, cystic fibrosis or chronic bronchitis.